Discovery Of Host Cell Candidate Proteins Critical To The Attachment By Cryptosporidium Sporozoites
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SUMMARYCryptosporidium is an AIDS-OI pathogen for which effective treatments are yet unavailable. It is also one ofthe top diarrheal-causing pathogens afflicting children in developing countries, causing significantly increasedmortality and stunted growth. Humans acquire cryptosporidial infection by ingesting oocysts, from whichsporozoites are released to infect gastrointestinal epithelial cells. The invasion of cryptosporidial sporozoitesis a first step to initialize the infection, but the key molecules in host cells interacting with the parasite duringthe invasion remain unknown.The long-term goal of this project is to elucidate the molecular mechanism regulating the cryptosporidialinvasion. Towards the goal, we initiated a UV irradiation-based host cell mutagenesis study, in which weproduced 43 host cell mutants derived from the parent wild-type (WT) HCT-8 cells and identified one of themutant (i.e., A05 mutant) was significantly defective in the parasite attachment and invasion. In comparison toWT cells, A05 cells are much less stretched out on the surface of culture plates, and cell-to-cell connectionsare generally loose, leaving apparent gaps between neighboring cells.Our central hypothesis is that a host cell membrane protein (or a protein complex) is required for theattachment and invasion by cryptosporidial sporozoites. The availability of A05 mutant makes it possible toidentify mutated and/or significantly regulated genes that can be prioritized for individually validation of theirrole in the parasite attachment and invasion. This R21 proposal represents an early stage of forward geneticstudies towards our long-term goal by achieving the following two specific aims.In aim 1, we will identify mutated and significantly regulated genes by transcriptomic and proteomic analyses.RNA-seq-based transcriptome analysis will be utilized to identity protein-coding genes in the A05 mutant thatare mutated and/or significantly regulated. Proteomic technology focus on identifying membrane proteins thatare mutated and/or significantly regulated. Identified genes/proteins will be prioritized for functional validation.In aim 2, we will validation of the function of candidate genes in the parasite attachment and invasion. Basedon the prioritized candidate genes in aim 1, their role in the attachment and invasion by sporozoites will beevaluated by antibody blocking assay, and by generating genetically modified cell lines from the WT HCT-8cells for testing their susceptibility to the attachment and invasion.This proposal focuses on identifying gene mutations associated with the resistance to the attachment andinvasion by Cryptosporidium sporozoites in A05 mutant for subsequent delineation of the molecularmechanism regulating the parasite attachment and invasion.
